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Metabolic Reprogramming of Pancreatic Ductal Adenocarcinoma Cells in Response to Chronic Low Ph Stress

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Metabolic Reprogramming of Pancreatic Ductal Adenocarcinoma Cells in Response to Chronic Low Ph Stress University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-15-2017 Metabolic Reprogramming of Pancreatic Ductal Adenocarcinoma Cells in Response to Chronic Low pH Stress Jaime Abrego University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Biochemistry Commons, Cancer Biology Commons, Molecular Biology Commons, and the Molecular, Genetic, and Biochemical Nutrition Commons Recommended Citation Abrego, Jaime, "Metabolic Reprogramming of Pancreatic Ductal Adenocarcinoma Cells in Response to Chronic Low pH Stress" (2017). Theses & Dissertations. 249. https://digitalcommons.unmc.edu/etd/249 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. Metabolic Reprogramming of Pancreatic Ductal Adenocarcinoma Cells in Response to Chronic Low pH Stress by Jaime Abrego A Dissertation Presented to the Faculty Of the University Of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor Of Philosophy Cancer Research Graduate Program Under the Supervision of Professor Pankaj K. Singh, Ph.D. University of Nebraska Medical Center Omaha, NE November, 2017 Supervisory Committee: Michael A. (Tony) Hollingsworth, Ph.D. Keith R. Johnson, Ph.D. Amarnath Natarajan, Ph.D. ii iii Acknowledgements First of all, I would like to thank my supervisor and mentor Dr. Pankaj K. Singh for his guidance, support, and motivation during the course of my PhD. I greatly appreciate his leadership, patience, and mentorship that have allowed me to become a better scientist over the years I worked under his tutelage. I am very thankful for the freedom and encouragement to pursue research projects fit to my scientific inquiry. For these reasons, as well as, all the learning experiences, I am indebted to him for giving me the opportunity to work in his lab and begin my journey to investigate the role of metabolism in cancer. I would also like to thank my supervisory committee members Dr. Michael A. (Tony) Hollingsworth, Dr. Keith R. Johnson, and Dr. Amarnath Natarajan for their excellent supervision, suggestions, and constructive criticism throughout my graduate career. Without their guidance, I would not have been able to identify and overcome many significant obstacles throughout my studies. I would also like to thank the current and former members of the Singh lab for their help in learning/optimizing laboratory techniques, as well as, for their moral and intellectual support throughout the years. I would like to especially acknowledge Dr. Venugopal Gunda for guiding my learning of metabolomics studies. The students and faculty of the Eppley Institute have made my personal endeavor through graduate school an enlightening and entertaining time. Moreover, I learned more than I could have ever imagined from my interactions with fellow students and faculty. Their kindness and generosity will always be cherished. Finally, I would like to thank friends and family for their unconditional love and support throughout my life and academic career. They have kept me focused and upbeat throughout the good times and the bad. Without their support and understanding, iv I would have never been able to complete my journey in graduate school. I would like to dedicate this thesis to my parents, Jaime & Irma Abrego, who brought my sisters and me to the United States from Mexico 13 years ago with the goal of giving us the opportunity for a brighter future. My dad works in a warehouse for over 50 hours per week and my mom works at a factory 4 days out of the week—and I cannot thank them enough for the work they have put to raise our family. They have raised us to be hard working, honest, and despite our economic limitations they’ve worked to give us the opportunity to attain higher education. It is because of them that I was able to conduct the research described in this thesis and for this reason this thesis belongs to them as well. v Metabolic Reprogramming of Pancreatic Ductal Adenocarcinoma Cells In Response To Chronic Low pH Stress Jaime Abrego, Ph.D. University of Nebraska Medical Center, 2017 Supervisor: Pankaj K. Singh, Ph.D. Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of all cancers with a 5-year survival rate of only 8.2%. This is because PDAC is diagnosed in its advanced stages and is characterized by radio and chemotherapy resistance. Aggressiveness of PDAC tumors is attributed to its high metabolic phenotype, which is characterized by increased glycolysis rate and lactate secretion, while oxidative metabolism is reduced. These metabolic features are required to fulfill the biosynthetic demands of proliferating PDAC cells. However, this increase in metabolic activity results in acidification of the extracellular space because the dense fibrotic stroma of PDAC tumors limits venting of protons into the vasculature thereby creating a chronic low pH microenvironment. Little is known regarding the physiology and metabolism of cancer cells enduring chronic low pH exposure. To demonstrate effects of low pH, PDAC cells were cultured in low pH 6.9~7.0 to establish chronic low pH as it occurs in tumors. These cells were compared to cells in physiological pH of 7.4, which is also the pH of cell culture, in order to evaluate physiological differences between these pH values. In these experiments, it was observed that cells in low pH have reduced clonogenic capacity and undergo a metabolic shift to oxidative metabolism that is supported by an increase in glutamine uptake. These observations exhibit a robust contrast to PDAC cells in control pH conditions that are highly glycolytic. Furthermore, in low pH there is increased vi transcription of the GOT1 enzyme, which mediates metabolic flux through the non- canonical glutamine metabolic pathway that allows synthesis of other metabolic substrates from glutamine. Upon shRNA-mediated depletion of GOT1, survival of PDAC cells in low pH was significantly impaired due to increase in ROS to cytotoxic levels. However, supplementing transfected clones with GOT1 metabolic product, oxaloacetate, resulted in growth rescue and reduction in ROS levels. Thus, in chronic low pH stress PDAC cells up-regulate non-canonical glutamine metabolism through increased transcription of GOT1, which allows PDAC cells to generate energy and metabolic co- factors to suppress cytotoxic ROS levels. Low pH is a universal feature of the PDAC tumor microenvironment and further dissection of metabolic adaptations to microenvironment conditions will result in more effective therapy for PDAC. vii Table of Contents Acknowledgements………………………….......................................................................iii Abstract……………............................................................................................................v Table of Contents……………...........................................................................................vii List of Figures……............................................................................................................ix List of Abbreviations……..................................................................................................xi Chapter 1: Introduction: The Evolution Of PDAC And Its Acidic Tumor Microenvironment…………………………….......................................................................1 Incidence of Pancreatic Ductal Adenocarcinoma..............................................................2 Development of Pancreatic Ductal Adenocarcinoma........................................................2 Features of Pancreatic Ductal Adenocarcinoma.............................................................14 Cellular Metabolism………………....................................................................................21 Cancer Cell Metabolism…………....................................................................................28 Tumor Microenvironment Acidification….........................................................................39 Hypothesis.……………....................................................................................................44 Figures Chapter 1……………..........................................................................................46 Tables Chapter 1……………...........................................................................................61 viii Chapter 2: Results: Got1-Mediated Anaplerotic Glutamine Metabolism Regulates Chronic Low pH Stress in PDAC Cells……………………………...................................64 Introduction………….......................................................................................................65 Materials & Methods…....................................................................................................67 Results…………..............................................................................................................76 Discussion………….........................................................................................................82 Chapter 2 Figures…........................................................................................................85 Chapter 3: Discussion: Conclusions & Future Directions.............................................103 Summary of Work…......................................................................................................104
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